Rewired m6A epitranscriptomic networks link mutant p53 to neoplastic transformation

N6-methyladenosine (m6A), one of the most prevalent mRNA modifications in eukaryotes, plays a critical role in modulating both biological and pathological processes. However, it is unknown whether mutant p53 neomorphic oncogenic functions exploit dysregulation of m6A epitranscriptomic networks. Here, we investigate Li-Fraumeni syndrome (LFS)-associated neoplastic transformation driven by mutant p53 in iPSC-derived astrocytes, the cell-of-origin of gliomas. We find that mutant p53 but not wild-type (WT) p53 physically interacts with SVIL to recruit the H3K4me3 methyltransferase MLL1 to activate the expression of m6A reader YTHDF2, culminating in an oncogenic phenotype. Aberrant YTHDF2 upregulation markedly hampers expression of multiple m6A-marked tumor-suppressing transcripts, including CDKN2B and SPOCK2, and induces oncogenic reprogramming. Mutant p53 neoplastic behaviors are significantly impaired by genetic depletion of YTHDF2 or by pharmacological inhibition using MLL1 complex inhibitors. Our study reveals how mutant p53 hijacks epigenetic and epitranscriptomic machinery to initiate gliomagenesis and suggests potential treatment strategies for LFS gliomas.

The authors have addressed most of the concerns and comments, and the addition of new experiments has significantly improved the manuscript. I only have two remaining concerns; 1. the authors show that high YTHDF2 expression correlates with poor prognosis in wtp53 glioma patients and not mutp53 patients -which questions the relevance of mutp53 in driving YTHDF2mediated oncogenic functions in vivo, and that this mechanism may not be the contributing factor for mutp53 GOF. 2. the lack of tumour formation in vivo using their model -however, you could claim that hyperplasia is a prelude to transformation but it is not an absolute proof -perhaps considering modifying the title of the manuscript?
Reviewer #2 (Remarks to the Author): The authors have conducted additional experiments and data analysis, and have thoroughly addressed our concerns. This is an interesting and innovative study. Thus, I would like to recommend the publication of this paper in Nature Communications.
Reviewer #3 (Remarks to the Author): The authors have addressed many of my concerns, and new results support the conclusions. However, the quality of some data should be further improved before publication. In particular: -To visualize the mutant p53/SVIL/MLL1 protein complex, the authors performed PLA in LFS astrocytes ( Fig. 4d). They should indicate: i) what the control is, ii) assess the effect of knockdown of mutp53, of SVIL, and of MLL1. They also performed coIP to demonstrate the SVIL-dependent formation of p53/SVIL/MLL1 complex. This experiment should include IgG and input controls -To demonstrate YTHDF2-dependent regulation of CDKN2B and SPOCK2, the authors performed immunofluorescence staining in LFS cerebral organoids engrafted in the mouse cortex ( Fig. 5j and Extended Data Fig. 6c). TO improve the quality of these data, I recommend to include, as controls, YTHDF2 staining and markers to distinguish the engrafted tissue.
-The annotation and legends of some figures should be improved to improve readability. For example, in Fig3, please explain the meaning of "peak" and "up" annotations.

Point-by-point response to reviewers' and editorial comments (Ms. No. NCOMMS-22-40872-T)
We thank all the reviewers for their positive assessment of our work and their highly constructive comments. This has helped us design new experiments and revise the manuscript to greatly improve the clarity and the impact of our findings on the field. Based on the reviewers' comments, we performed new experiments, including 4 new figures (Fig. 4e, f and Supplementary Fig. 6c, d) and 3 revised figures (Fig. 3c, d, 4d, and 5j), and revised the manuscript accordingly. We trust that all comments have been addressed satisfactorily either through additional experiments or through better explanations to clarify specific points.

Reviewers' Comments:
Reviewer #1: 1. The authors show that high YTHDF2 expression correlates with poor prognosis in wtp53 glioma patients and not mutp53 patients -which questions the relevance of mutp53 in driving YTHDF2mediated oncogenic functions in vivo, and that this mechanism may not be the contributing factor for mutp53 GOF.
Response: Thank you for this comment. Our results indeed demonstrate that YTHDF2 functions as an oncoprotein in glioma development and its expression is correlated to poor prognosis (Fig.  2f). Mutant p53 regulates the YTHDF2-associated m 6 A epitranscriptome, which contributes to glioma initiation and progression. In p53 mutant glioma specimens, YTHDF2 expression is at a relatively high level due to mutant p53-mediated YTHDF2 transcription upregulation (Fig 7b and  Supplementary Fig. 8a, TCGA glioma data). Therefore, the additional changes of YTHDF2 expression (moderately high vs high) in p53 mutant glioma patients only have limited effects on patient survival since elevated YTHDF2 by mutant p53 is a sufficient dominant influencer of patient survival among p53 mutant glioma patients. To make it clear, we revised our conclusion to "Interestingly, high expression of YTHDF2 was associated with poor prognosis in glioma patients with wild-type p53 but not mutant p53 (Fig. 2g), suggesting that high YTHDF2 expression is correlated to poor prognosis in p53 wild-type glioma patients and the elevated level of YTHDF2 by mutant p53 is a sufficient dominant influencer of patient survival among p53 mutant glioma patients." (lines 239-241 of the revised manuscript) 2. The lack of tumour formation in vivo using their model -however, you could claim that hyperplasia is a prelude to transformation but it is not an absolute proof -perhaps considering modifying the title of the manuscript?